Process for producing amide compound using microbial catalyst

ABSTRACT

This invention relates to a process for producing an amide compound from a nitrile compound using a microbial catalyst, wherein a microbial cell having nitrile hydratase activity of 50 U or higher per mg of dry cell at a reaction temperature of 10° C. is brought into contact with a nitrile compound in an aqueous medium without being immobilized. This method utilizes a microbial cell that exhibits high nitrile hydratase activity in the reaction without being entrap-immobilized. Thus, an amide compound can be effectively produced from a nitrile compound without problems of decreased reaction speed or lowered amount produced per unit cell amount, which are caused by entrap-immobilization. Accordingly, an amide compound can be produced within a very short period of time in the case of a batch reaction and with a very small-scale facility in the case of a continuous reaction.

TECHNICAL FIELD

[0001] The present invention relates to a process for producing an amidecompound from a nitrile compound using a microorganism having nitrilehydratase activity.

BACKGROUND ART

[0002] Recently, a process for synthesizing a compound using abiocatalyst has been used for producing a variety of compounds becauseof advantages such as moderate reaction conditions, simplified reactionprocesses, and high purity of reaction products due to small amounts ofby-products.

[0003] Since the discovery of nitrile hydratase, an enzyme that convertsa nitrile compound into an amide compound, biocatalyst utilization hasbeen actively studied in the production of amide compounds (JP PatentPublication (Kokai) No. 11-123098, JP Patent Publication (Kokai) No.7-265091, JP Patent Publication (Kokoku) No. 56-38118, and JP PatentPublication (Kokai) No. 11-89575).

[0004] At present, a microorganism having nitrile hydratase activity isused for producing acrylamide, nicotineamide, or the like at theindustrial level for a superior reaction process from the viewpoints ofoperability, safety, economic efficiency, and other factors.

[0005] Up to the present, a considerable number of microorganisms havebeen found having nitrile hydratase activities. Examples thereof includemicroorganisms belonging to the genera Nocardia, Corynebacterium,Bacillus, Pseudomonas, Micrococcus, Rhodococcus, Acinetobacter,Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter,Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium, andPseudonocardia.

[0006] Among them, the genera Pseudomonas, Bacillus, Rhodococcus, andPseudonocardia express a nitrile hydratase having a very high level ofactivity and stability. Thus, they are used at the industrial level orlevels similar thereto.

[0007] Further, when culturing these microorganisms, it is known that amicrobial cell having highly active nitrile hydratase is obtained by amethod of adding nitriles or amides (JP Patent Publication (Kokoku) Nos.61-43996 and 61-43999), a method of adding amino acid (JP PatentPublication (Kokoku) Nos. 61-43997 and 61-43998), a method of addingsome kind of metal ion (JP Patent Publication (Kokai) No. 61-162193, JPPatent Publication (Kokoku) No. 6-55148, and JP Patent Publication(Kokai) No. 8-187092), or the like.

[0008] In contrast, a biocatalyst has low stability with regard to heat,and thus, reactions must be carried out at low temperatures. Thisresults in a decreased reaction speed per catalyst. When producing acompound using a biocatalyst at the industrial level, therefore, thecatalyst concentration in the reaction tank should be raised.

[0009] A currently known industrial process for producing an amidecompound from a nitrile compound using a biocatalyst is similarlycarried out by immobilizing microbial cells to make them particulate,raising the catalyst concentration in the reaction tank, andfacilitating catalyst separation (see Kagaku to Kogyo (Chemistry andChemical Industry) Vol. 43, No. 7, p. 1098-1101 (1990), JP PatentPublication (Kokai) Nos. 54-143593 and 54-144889). Also, a method forimmobilizing the cell is studied (see JP Patent Publication (Kokai) Nos.57-39792 and 62-294083). When effective production of an amide compoundat the industrial level is intended, immobilization of cells at a higherconcentration has been considered to be important (see JP PatentPublication (Kokai) No. 7-203964).

[0010] However, the present inventors entrap-immobilized a microbialcell that exhibits high nitrile hydratase activity and used it in thereaction. As a result, it was confirmed that a nitrile compound as areaction substrate and/or an amide compound as a reaction product causeddiffusion defects in the entrap-immobilized particles, and the reactionspeed was significantly decreased.

[0011] For example, according to the comparison of initial reactionspeeds between the entrap-immobilized cell and the unimmobilized cell,the reaction speed was significantly decreased to one-tenth or lower,depending on reaction conditions. Not only the initial reaction speed issignificantly decreased, but also the activity of the enzyme in theentrap-immobilized catalyst, which does not fully contribute to thereaction due to diffusion defect, is lowered during the reaction. Thisalso lowers the amount of amide compound produced per unit cell amount.

[0012] Specifically, decreased reaction speed or lowered amount of amidecompound produced per unit cell amount as mentioned above results inunfavorable conditions. Under such conditions, it takes a long time toaccumulate the targeted amount when producing an amide compound by abatch reaction, and the size of facilities must be enlarged in the caseof a continuous reaction.

[0013] Accordingly, an object of the present invention is to solveproblems occurring in the process for producing an amide compound from anitrile compound using a biocatalyst, such as decreased reaction speedor lowered amount of amide compound produced per unit cell amount. Theseproblems are caused by the use of immobilized microbial cell with highlyexhibited nitrile hydratase activity.

[0014] In order to attain the above object, the present inventors haveconducted concentrated studies concerning a more suitable form ofcatalyst than the immobilized catalyst. As a result, they have foundthat an amide compound could be more effectively produced when using amicrobial cell that exhibits high nitrile hydratase activity of 50 U orhigher per mg of dry cell at 10° C., and bringing the microbial cellinto contact with a nitrite compound while suspended in an aqueousmedium, than is the case when immobilizing the cell. This has led to thecompletion of the present invention.

[0015] More specifically, the present invention relates to a process forproducing an amide compound from a nitrile compound using a microbialcatalyst, wherein a microbial cell having nitrile hydratase activity of50 U or higher per mg of dry cell at a reaction temperature of 10° C. isbrought into contact with a nitrile compound in an aqueous mediumwithout being immobilized.

DISCLOSURE OF THE INVENTION

[0016] The present invention is hereafter described in detail.

[0017] In the present invention, any microorganisms may be used as longas they have nitrile hydratase activity of 50 U or higher per mg of drycell at a reaction temperature of 10° C. Examples of preferablemicroorganisms include those belonging to the genera Bacillus,Bacteridium, Micrococcus, Brevibacterium (JP Patent Publication (Kokoku)No. 62-21519), Corynebacterium, Nocardia (JP Patent Publication (Kokoku)No. 56-17918), Pseudomonas (JP Patent Publication (Kokoku) No.59-37951), Microbacterium (JP Patent Publication (Kokoku) No. 4-4873),Rhodococcus (JP Patent Publication (Kokoku) Nos. 4-4873, 6-55148, and7-40948), Achromobacter (JP Patent Publication (Kokai) No. 6-225780),and Pseudonocardia (JP Patent Publication (Kokai) No. 9-275978).Bacteria of the genus Rhodococcus are more preferable.

[0018] Alternatively, a transformant may be used. This is prepared byobtaining the aforementioned microorganism-derived nitrile hydratasegene and introducing the gene as such, or an artificially modified formthereof, into an arbitrary host.

[0019] Examples of the transformants include E. coli MT 10770 (FERMP-14756) transformed with a nitrile hydratase of the genus Achromobacter(JP Patent Publication (Kokai) No. 8-266277), E. coli MT 10822 (FERMBP-5785) transformed with a nitrile hydratase of the genusPseudonocardia (JP Patent Publication (Kokai) No. 9-275978), or amicroorganism transformed with a nitrile hydratase of the genusRhodococcus rhodochrous (JP Patent Publication (Kokai) No. 4-211379).

[0020] The unit “U” of enzyme activity used in the present inventionmeans that 1 μmol of corresponding amide compound is generated from anitrile compound per minute. The term “enzyme activity” used hereinrefers to the value of enzyme activity measured utilizing a nitrilecompound that is used in production.

[0021] The enzyme activity is measured by placing 5 mL of 50 mMphosphate buffer adjusted to the optimal pH (e.g., pH 7) of the enzymein a test tube having a diameter of 30 mm, suspending 2 mg of thecultured and washed cells (dry weight) therein, and shaking the tube ina water tank at 10° C. About 5 minutes later, a phosphate bufferprepared in advance containing 1 to 5% of nitrile compound, placed at10° C. and adjusted to the optimal pH, is added. The concentration ofthe amide compound generated after an arbitrary reaction time ismeasured using analyzing equipment such as gas chromatography or liquidchromatography, thereby calculating the enzyme activity.

[0022] The reaction time is determined in such a manner that a reactionsolution retains a nitrile compound of a concentration at which thereaction speed is not decreased, and the concentration of the amidecompound generated is high enough to be accurately measured at the endof the reaction.

[0023] The present invention is effective when a microbial cell havingenzyme activity of 50 U or higher per mg of dry cell at 10° C. is used.It is more effective with the use of a microbial cell having enzymeactivity of 80 U or higher, and even more effective with activity of 100U or higher.

[0024] The nitrile compound according to the present invention isconverted into a corresponding amide compound through the action of anitrile hydratase. Examples thereof include: aliphatic saturatednitriles as exemplified by acetonitrile, propionitrile, succinonitrile,and adiponitrile; aliphatic unsaturated nitriles as exemplified byacrylonitrile and methacrylonitrile; aromatic nitriles as exemplified bybenzonitrile and phthalodinitrile; and heterocyclic nitriles asexemplified by 3-cyanopyridine and 2-cyanopyridine. Because of thechemical and physical properties of a nitrile compound, the substratespecificity of a nitrile hydratase enzyme, and the industrial point ofview, acrylonitrile and cyanopyridine are preferable as target compoundsof the present invention.

[0025] In the present invention, a form of catalyst without beingentrap-immobilized is such that a membrane of a microbial cell is in adirect contact with a reaction solution. An example thereof is a form ofcatalyst treated in an entrap-immobilization method in which a cell isnot entrapped with high molecular substances such as polyacrylamide,polyvinyl alcohol, carrageenan, agar, gelatin, or alginic acid.

[0026] More specifically, the “catalyst without beingentrap-immobilized” according to the present invention is a microbialcell itself that was cultured and optionally subjected to washing orother forms of treatment, a microbial cell that was chemically treatedwith a substance having a polyfunctional group such as glutaraldehyde,or a microbial cell that was chemically bonded onto a surface of a glassbead, resin, silica gel, or the like.

[0027] The use of a cell chemically treated with glutaraldehyde as acatalyst is particularly preferable from the viewpoint of improvement inthe stability of catalyst enzyme activity.

[0028] The operation that brings a microbial cell into contact with anitrile compound in an aqueous medium refers to one in which a microbialcell having nitrile hydratase activity is brought into contact with anitrile compound in water or in an aqueous medium prepared bydissolving, for example, a stabilizer for ion strength, pH buffercapacity, or nitrile hydratase activity in water. This may be carriedout by a batch system or a continuous system. A form of reaction isselected depending on properties of reaction substrate, reactionsolution, target compound, and the like, or scale of production, and areaction apparatus is designed based thereon.

[0029] Preferably, reaction conditions such as reaction temperature andpH are controlled to be optimal so that an amide compound can beproduced on a smaller scale or within a shorter time.

[0030] The concentration of the amide compound accumulated by the abovemethod is preferably 20% or higher, and more preferably 50% or higher,from an industrial point of view.

[0031] This description includes part or all of the content as disclosedin the description of Japanese Patent Application No. 2000-387537, whichis a priority document of the present application.

BEST MODES FOR CARRYING OUT THE INVENTION

[0032] The present invention is hereafter described in more detail withreference to the following examples, although it is not limited thereto.In the following examples, “%” is by mass unless otherwise specified.

EXAMPLE 1 Production of Acrylamide Using a Microbial Cell in an AqueousMedium

[0033] (1) Culture of Cell

[0034] (i) Conditions for Preculture:

[0035] (Composition of Medium)

[0036] 2% fructose, 5% polypeptone (Nihon Pharmaceutical Co., Ltd.),0.3% yeast extract (Oriental Yeast Co., Ltd.), 0.1% KH₂PO₄, 0.1% K₂HPO₄,0.1% MgSO₄.7H₂O, pH 7

[0037] (Culture Method)

[0038] A medium (100 ml) was fractionated into a 500 ml conical flask,the flask was cotton plugged, and it was then sterilized in an autoclaveat 121° C. for 20 minutes. Rhodococcus rhodochrous J1 (FERM BP-1478) wasinoculated and subjected to shake culture at 30° C. for 48 hours.

[0039] (ii) Conditions for Main Culture:

[0040] (Composition of Medium)

[0041] Initial medium: 0.2% yeast extract, 0.1% KH₂PO₄, 0.1% K₂HPO₄,0.1% MgSO₄.7H₂O, 0.002% CoCl₂.6H₂O, 0.025% ammonium sulfate, 2%fructose, 2% urea, 0.4% ethanol, 0.1% Pluronic L61 (Asahi Denka Co.,Ltd.), pH 7

[0042] Medium added later: 20% fructose, 5% ethanol, 6% ammoniumsulfate, pH 6.5

[0043] (Culture Method)

[0044] The initial medium (2 liters) was fractionated into a 3-litermini-jar fermenter and sterilized in an autoclave at 121° C. for 20minutes. Separately, fructose, ethanol, and urea were asepticallyfiltered using a 0.45 micron filter paper (Advantec Toyo Kaisha, Ltd.)and added to the medium.

[0045] Culture was conducted under conditions at a pressure inside thetank of 0.098 MPa, an agitation rate of 600 rpm, an air-flow rate of 1vvm, a pH of 7, and a temperature of 30° C. Culture was terminated whenthe maximal enzyme activity occurred. Thereafter, the culture productwas washed with a 50 mM phosphate buffer (pH 7.7), and a suspension ofcell (weight of dry cell: 15%) was obtained.

[0046] (2) Measurement of Nitrile Hydratase Activity

[0047] A 50 mM phosphate buffer (4.98 ml, pH 7.7) and 20 μL ofsuspension of cell were added and mixed in a test tube having a diameterof 30 mm, and the tube was shaken in a tank at 10° C. for 5 minutes. A50 mM phosphate buffer (5 mL, pH 7.7) containing 5.0% acrylonitrile,which was previously set at 10° C., was added thereto, the product wasallowed to react for 10 minutes, the cells were separated by filtration,and acrylamide generated was quantified by gas chromatography (GC-14B,SHIMADZU CORPORATION). Analysis was conducted using a 1 m glass columnfilled with Parabox PS (a column filler, Waters) at a column temperatureof 230° C., and the FID detection was conducted at 250° C. The resultindicated that 1.2% of acrylamide was generated. When “1 U” is definedas an amount of activity resulting when 1 micromole of acrylonitrile isconverted into acrylamide at a reaction temperature of 10° C. within areaction time of 1 minute, the activity of the cell for convertingacrylonitrile into acrylamide was 56 U per mg of dry cell at 10° C.

[0048] (3) Conversion of Acrylonitrile into Acrylamide

[0049] A 50 mM TRIS (2-amino-2-hydroxymethyl-1,3-propanediol)hydrochloride buffer (664 g, pH 7.7) was placed in a 1 liter-jacketedseparable flask. The suspension of cell obtained above was added theretoso as to bring the weight of dry cell to 90 mg. Acrylonitrile wascontinuously added thereto in order to maintain the acrylonitrileconcentration at 2% at an agitation rate of 180 rpm at 18° C.

[0050] As a result, the concentration of the acrylamide, which wasproduced 25 hours after the initiation of acrylonitrile addition,reached the target level of 45%.

COMPARATIVE EXAMPLE 1 Production of Acrylamide Using anEntrap-Immobilized Microbial Cell

[0051] (1) Immobilization of Cells

[0052] The suspension of cell obtained in Example 1 having activity of56 U in terms of converting acrylonitrile into acrylamide was added toan equivalent amount of an aqueous solution of thoroughly dissolved 3%sodium alginate (Kanto Kagaku), and they were thoroughly mixed. Thismixture was added dropwise to an aqueous solution of 1M calcium chloridethrough a silicon tube having an inner diameter of 2 mm. Thus, particlesof immobilized cell having particle diameters of about 3 mm wereobtained. The particles of immobilized cell were washed with a 50 mMTRIS hydrochloride buffer (adjusted to pH 7.7) to obtain immobilizedcells.

[0053] (2) Conversion of Acrylonitrile into Acrylamide

[0054] A 50 mM TRIS hydrochloride buffer (664 g, pH 7.7) was placed in a1 liter-jacketed separable flask. The immobilized cells obtained abovewere added thereto so as to bring the weight of dry cell to 90 mg.Acrylonitrile was continuously added thereto in order to maintain theacrylonitrile concentration at 2% at an agitation rate of 180 rpm at 18°C.

[0055] As a result, the acrylamide concentration did not reach thetarget level of 45% even 50 hours after the initiation of acrylonitrileaddition.

EXAMPLE 2 Production of Acrylamide Using Microbial Cell in an AqueousMedium

[0056] (1) Culture of Cell

[0057]Pseudomonas chlororaphis B23 (FERM BP-187) cell was cultured inthe manner as described in the Example of JP Patent Publication (Kokai)No. 2-177883. The activity of this cell for converting acrylonitrileinto acrylamide was measured in the same manner as in Example 1 at pH7.7. As a result, the activity was 90 U per mg of dry cell at 10° C.

[0058] (2) Conversion of Acrylonitrile into Acrylamide

[0059] A 50 mM phosphate buffer (850 mL, pH 7.7) and 0.4 g of cell (on adry basis) were added to a jacketed separable flask (internal volume: 1liter). The reaction was carried out by continuously addingacrylonitrile while stirring at 3° C. to maintain the acrylonitrileconcentration at 2%.

[0060] The acrylamide concentration reached the target level of 20%three hours later.

COMPARATIVE EXAMPLE 2 Production of Acrylamide using Entrap-ImmobilizedMicrobial Cell

[0061] (1) Entrap-Immobilization of Cell

[0062] An aqueous solution of monomer mixture was prepared so as tocomprise 30%, 1%, and 4% of acrylamide, methylenebisacrylamide, and2-dimethylaminopropyl methacrylamide, respectively.

[0063] Subsequently, the suspension of cell having activity of 90 U interms of converting acrylonitrile into acrylamide obtained in Example 2,an aqueous monomer solution, an aqueous solution of 10%N,N,N′,N′-tetramethyl ethylene diamine, and an aqueous solution of 10%ammonium persulfate were subjected to line-mixing at a mixing ratio of50:20:1:1. The effluents were successively placed on a bat having a sizeof 300×300×30 mm and then polymerized thereon.

[0064] The produced cell-immobilized gel sheet was cut into small piecesof about 0.5 mm² using a knife to obtain particles of acrylamide polymerentrap-immobilized cell. The particles of immobilized cell were washedby dipping in an aqueous solution of 0.1% sodium acrylate (adjusted topH 7 with the aid of sodium hydroxide) for preparation.

[0065] (2) Conversion of Acrylonitrile into Acrylamide

[0066] Acrylonitrile was converted into acrylamide using the method andthe apparatus as described in Example 2.

[0067] The acrylamide concentration did not reach the target level of20% eight hours later.

COMPARATIVE EXAMPLE 3 Production of Acrylamide using a Microbial CellHaving Low Nitrile Hydratase Activity

[0068] (1) Culture of Cell and Preparation of Catalyst

[0069] In the same manner as in Example 1, the Rhodococcus rhodochrousJ1 (FERM BP-1478) cell was cultured. When the activity of the cell forconverting acrylonitrile into acrylamide, which was measured based onthe method for measuring activity as described in Example 1, reached 20U per mg of dry cell at 10° C., culture was terminated. Thereafter, theculture product was washed with a 50 mM phosphate buffer (pH 7.7), and asuspension of cell (weight of dry cell: 15%) was obtained.

[0070] (2) Conversion of Acrylonitrile into Acrylamide

[0071] In accordance with the method described in Comparative Example 2,a suspension of acrylamide polymer entrap-immobilized cell was firstprepared.

[0072] Subsequently, the aforementioned suspension of immobilized cellor suspension of unimmobilized cell was used as a microorganism in anamount of 225 mg in terms of the weight of dry cell, thereby convertingacrylonitrile into acrylyamide. As a result, the acrylamideconcentration reached the target level of 45% about 100 hours later withthe use of a suspension of either immobilized or unimmobilized cells.

[0073] Specifically, there was no significant difference betweenimmobilized cells and unimmobilized cells when the cell exhibitedactivity of 20 U.

[0074] All publications, patents, and patent applications cited hereinare incorporated herein by reference in their entirety.

[0075] Industrial Applicability

[0076] In the method according to the present invention, a microbialcell that exhibits high nitrile hydratase activity is used in thereaction without being entrap-immobilized. Thus, an amide compound canbe effectively produced from a nitrile compound without problems ofdecreased reaction speed or lower amount produced per unit cell amount,which are caused by entrap-immobilization. Accordingly, an amidecompound can be produced within a very short period of time in the caseof a batch reaction and with a very small-scale facility in the case ofa continuous reaction.

What is claimed is:
 1. A process for producing an amide compound from anitrile compound using a microbial catalyst, wherein a microbial cellhaving nitrile hydratase activity of 50 U or higher per mg of dry cellat a reaction temperature of 10° C. is brought into contact with anitrile compound in an aqueous medium without being entrap-immobilized.2. The process for producing an amide compound according to claim 1,wherein the microbial cell having nitrile hydratase activity of 50 U orhigher per mg of dry cell at a reaction temperature of 10° C. is atleast one member selected from the group consisting of the generaNocardia, Corynebacterium, Bacillus, Pseudomonas, Micrococcus,Rhodococcus, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium,Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter,Achromobacter, Agrobacterium, and Pseudonocardia.
 3. The process forproducing an amide compound according to claim 1, wherein the microbialcell having nitrile hydratase activity of 50 U or higher per mg of drycell at a reaction temperature of 10° C. is a transgenic microorganism,which expresses a nitrile hydratase gene of at least one microorganismselected from the group consisting of the genera Nocardia,Corynebacterium, Bacillus, Pseudomonas, Micrococcus, Rhodococcus,Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella,Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter,Agrobacterium, and Pseudonocardia.
 4. The process for producing an amidecompound according to any one of claims 1 to 3, wherein the nitrilecompound is acrylonitrile or cyanopyridine.